During early mouse development, an extraembryonic cell type known as the distal visceral endoderm (DVE) plays a central role in the anterior-posterior (A-P) patterning of the embryo. Recent studies have shown that the signaling pathway for the Transforming Growth Factor-beta (TGF) ligand Nodal is essential for formation of both the visceral endoderm and the DVE, as well as the early specification of A-P asymmetry at peri-implantation stages. In this competing renewal application, we propose to continue our long-standing studies of the regulation of Nodal pathway activity and its role in patterning the mouse embryo. In the previous and current funding periods, we have shown that the activity of the Nodal pathway during mouse development is tightly controlled by the activities of the EGF-CFC co-receptors, Cripto and Cryptic. In our preliminary studies, we demonstrate that the complete absence of EGF-CFC function in Cripto; Cryptic double mutants leads to a severe pre-gastrulation lethal phenotype that includes defects in formation of the visceral endoderm and DVE, and the absence of detectable A-P asymmetry. We also show that up-regulation of Nodal pathway activity in culture can lead primitive endoderm stem (XEN) cells to contribute to visceral endoderm in vivo. Based on these findings, we hypothesize that the Nodal signaling pathway is essential for specification of the A-P axis and differentiation of the visceral endoderm during peri-implantation development, and that EGF-CFC proteins are required for modulating this pathway activity. Our proposed studies will investigate these EGF-CFC functions using a synergistic combination of in vivo, ex vivo, cell culture, and biochemical analyses to pursue the following three specific aims: 1) Investigation of EGF-CFC and Nodal pathway function in visceral endoderm formation through studies of Cripto and Cryptic expression, analyses of visceral endoderm defects in EGF-CFC mutants, and studies of XEN cell differentiation. 2) Investigation of EGF-CFC and Nodal pathway function in the generation of A-P asymmetry by determining the requirements for EGF-CFC function in the earliest known events in A-P asymmetry, analyzing the timing of this requirement, and lineage-marking of primitive endoderm sub-populations. 3) Analysis of EGF- CFC function as co-receptors for Nodal by examining the molecular regulation of Nodal precursor processing and activity, and by analyzing the significance of their GPI-linkage. Project narrative: Our proposed studies will address central questions about the initial cell fate decisions and anterior- posterior patterning of the mammalian embryo. These studies of early embryonic development should be important for understanding the high rate of embryo loss during early human pregnancy, and may improve the success rate of in vitro fertilization techniques. In addition, understanding the molecular regulation of stem cells derived from the early embryo, such as primitive endoderm stem cells, will be significant for the efficient directed differentiation of stem cells into desired cell types for potential use in stem cell-based therapies.